Capasso et al. BMC Molecular and Cell Biology (2021) 22:18 BMC Molecular and https://doi.org/10.1186/s12860-021-00353-x Cell Biology

RESEARCH ARTICLE Open Access Intracellular pH regulation: characterization and functional investigation of H+ transporters in pistillata Laura Capasso1,2, Philippe Ganot1, Víctor Planas-Bielsa1, Sylvie Tambutté1 and Didier Zoccola1*

Abstract Background: Reef-building regularly experience changes in intra- and extracellular H+ concentrations ([H+]) due to physiological and environmental processes. Stringent control of [H+] is required to maintain the homeostatic acid-base balance in cells and is achieved through the regulation of intracellular pH (pHi). This task is especially challenging for reef-building corals that share an endosymbiotic relationship with photosynthetic dinoflagellates (family Symbiodinaceae), which significantly affect the pHi of coral cells. Despite their importance, the pH regulatory proteins involved in the homeostatic acid-base balance have been scarcely investigated in corals. Here, we report in the coral Stylophora pistillata a full characterization of the genomic structure, domain topology and phylogeny of three major H+ transporter families that are known to play a role in the intracellular pH regulation of cells; we investigated their tissue-specific expression patterns and assessed the effect of seawater acidification on their expression levels. Results: We identified members of the Na+/H+ exchanger (SLC9), vacuolar-type electrogenic H+-ATP hydrolase (V- ATPase) and voltage-gated proton channel (HvCN) families in the genome and transcriptome of S. pistillata.In addition, we identified a novel member of the HvCN gene family in the cnidarian subclass Hexacorallia that has not been previously described in any . We also identified key residues that contribute to H+ transporter substrate specificity, protein function and regulation. Last, we demonstrated that some of these proteins have different tissue expression patterns, and most are unaffected by exposure to seawater acidification. Conclusions: In this study, we provide the first characterization of H+ transporters that might contribute to the homeostatic acid-base balance in coral cells. This work will enrich the knowledge of the basic aspects of coral biology and has important implications for our understanding of how corals regulate their intracellular environment. Keywords: H+ transport, Reef-building corals, pH regulation, Gene expression, Ocean acidification

* Correspondence: [email protected] 1Centre Scientifique de Monaco, 8 quai Antoine 1er, 98000 Monaco, Monaco Full list of author information is available at the end of the article

© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Capasso et al. BMC Molecular and Cell Biology (2021) 22:18 Page 2 of 19

Background through acid-base transport mechanisms [19]. Over the Coral reefs are among the most valuable ecosystems on previous years, a number of acid-base cellular sensors, earth, harbouring more than one-third of ocean bio- e.g., the acid-base sensing enzyme soluble adenylyl cy- diversity and providing economic benefits to tropical clase (sAC), and transport proteins have been proposed coastal nations worldwide [1]. Scleractinian corals are in corals on the basis of those characterized in verte- the major constructors of coral reefs, and most of them brates [20–22]. Acid-base transport proteins, which con- − have mutualistic relationships with endosymbiotic dino- trol pHi regulation, fall into two groups, namely, HCO3 flagellate (family Symbiodinaceae)-symbiotic corals that (solute carrier-SLC transporter family 4 and 26) and H+ provide photosynthetic products that support coral me- membrane transporters [23]. Based on the energy source tabolism, growth and reproduction [2–4]. Despite their used for H+ extrusion, H+ membrane transporters can environmental significance, scleractinian corals face be further divided into transporters, pumps and chan- many challenges to their survival, including ocean acid- nels. Transporters can move H+ against their electro- ification (OA), as a result of rising carbon dioxide levels chemical gradient by coupling H+ transport with pre- in the atmosphere [5]. The effects of OA on the coral existing ion gradients as an energy source. This group calcification rate and skeletal growth, including varied includes the SLC9 family, also known as Na+/H+ ex- species-specific responses, have been previously docu- changers, which harness the electrochemical gradient of mented [6–9]. Most of these effects have been proposed Na+ maintained by Na+/K+-ATPase [24]. The SLC9 fam- to be linked to acid-base regulatory processes and to ily can be further divided into three subfamilies: subfam- lead to altered ionic concentration and energy expend- ily A 1–9 (cation proton antiporter 1, CPA1), subfamily iture and allocation [10]. Although acid-base regulatory B1–2 (cation proton antiporter 2, CPA2) and subfamily processes might modulate physiological responses to C1–2 (Na-transporting carboxylic acid decarboxylase, OA, the pH regulatory proteins responsible for acid-base NaT-DC) [25]. The second group of H+ membrane + homeostasis are still poorly characterized in corals. transporters in charge of pHi regulation includes H Corals are diploblastic , which means that they pumps that allow H+ to move against its concentration are made of two cell layers, an ectoderm and an endo- gradient by coupling H+ transport to ATP hydrolysis. derm, separated by a layer of mesoglea. Both ectoderm Vacuolar-type electrogenic H+-ATP hydrolases (V- + and endoderm are present in the oral and aboral tissue ATPases), which transport H via V0 V-ATPase subunit- located on either side of the gastrovascular cavity (coe- a[26], and plasma membrane Ca+2-ATPase (PMCA), lenteron) [11]. Each tissue possesses several cell subtypes which extrudes Ca+2 in exchange for H+ [19, 27, 28], that achieve acid-base homeostasis via pH regulation belong to this group. Although some studies claim within physiological boundaries compatible with cell that pHi regulation is not the primary role of these functioning [12]. This is especially challenging for sym- H+ pumps in most mammalian cells [19], others have biotic corals, as photosynthesis significantly increases the suggested the opposite [29–32]. Members of the last pH of coral cells [13, 14]. Symbiotic corals can experi- group, the H+ channels, allow H+ to passively diffuse ence large variations in extracellular pH (pHe) due to down its electrochemical gradient whenever the regu- physiological (e.g., respiration, calcification and photosyn- latory gate is open and includes voltage-gated proton thesis) and environmental (e.g., metabolism of reef- channels (HvCN) [33, 34]. associated organisms, tides, water flow, upwelling and Despite their fundamental importance in pHi regula- ocean acidification) factors. For example, symbiont photo- tion, the investigation of these H+ transporters is incom- synthesis and respiration of both the host and symbiont plete and includes only a limited number of marine drive wide pHe variations (from pH 8.5 to 6) in the in- species. For example, SLC9 and V-ATPase proteins have ternal fluid of the coelenteron [13–18], and exposure to been identified in the transcriptome of calcifying pri- acidified seawater decreases the pHe in the extracellular mary mesenchymal cells of the sea urchin [35] and in calcifying medium (ECM) [9], where calcification occurs. the gills of fishes, where they are believed to play a role These pHe variations can also have an impact on the regu- in acid-base regulation of the branchial epithelium [36, lation of intracellular pH (pHi). For example, decreases in 37]. In addition, in white shrimp (Litopenaeus vanna- the pHe of the ECM (from 8.3 to 7.8) under acidified sea- mei), squid (Sepioteuthis lessoniana), and sea anemone water conditions affect the pHi of calicoblastic cells. Des- (Anemonia viridis), SLC9 has been shown to play a role pite these variations, coral cells are able to maintain their in the response to low-pH stress [38–40]. In corals, pHi within narrow limits (7.1–7.4) [9], showing that corals among the acid-base transport proteins in charge of pHi − possess efficient pHi regulatory mechanisms that account regulation, only HCO3 transporters have been charac- for this stability. terized [20], whereas information regarding H+ mem- +2 Under pHi stress, an appropriate response depends on brane transporters is limited to Ca -ATPase [41] and the ability to sense acid-base disturbances and react V1 V-ATPase subunit B [42]. Capasso et al. BMC Molecular and Cell Biology (2021) 22:18 Page 3 of 19

In the present study, we provide a full characterization the percentages of similarity and identity varied for each of the genomic structure, domain topology and phyl- subfamily. For members of subfamily A, the percentage of ogeny of the principal H+ transporters that might be similarity varies between 50 and 57% (sharing 35% iden- involved in the intracellular pH regulation and mainten- tity). However, members of subfamily B share 78% similar- ance of cellular physiological homeostasis in the symbi- ity (sharing 62% identity). spiSLC9 proteins also exhibit otic coral Stylophora pistillata. These transporters similarity with hSLC9s: spiSLC9A has 42–55% identity include Na+/H+ exchangers, which are ion transporters and 60–71% similarity to hSLC9A; spiSLC9B has 43–47% that concurrently transport Na+ into the cell and H+ out identity and 61–66% similarity to hSLC9B; and spiSLC9C + of the cell; voltage-gated H channels (HvCN), which has 26% identity and 50% similarity to hSLC9C. In represent a specific subset of proton channels that have addition, spiSLC9s possess conserved features with voltage- and time-dependent gating and thus only open hSLC9s, including residues related to Na+/H+ exchanger + to extrude H from the cell; and V0 V-ATPase subunit- activity, such as F161, P167–168, R440 and G455–456, for a, which connects the two portions (V0 and V1) of the members of subfamily A (Additional file 4); glycine zipper multi-subunit enzyme V-ATPase and is crucial for pro- sequences (SLC9B1-GZ1: 275–283; SLC9B2-GZ1: 184– ton transport [19, 30, 43–45]. Furthermore, we charac- 192; GZ2: 210–216; GZ3: 263–271) for members of sub- terized the gene expression patterns of H+ transporters family B (Additional file 5); and a conserved voltage- to determine whether they are differentially expressed in sensing domain (VSD) composed of four transmembrane coral tissues, and we discussed their potential physio- segments S1-S4 and a cyclic nucleotide-binding domain logical roles. In addition, we investigated the molecular (CNBD) for members of subfamily C (Additional file 6). response of S. pistillata to ocean acidification by asses- Regarding post-translational modifications, several sing the effect of external seawater acidification on the phosphorylation and N-glycosylation sites were pre- levels of H+ transporter expression after 1 week and 1 dicted for spiSLC9s (Additional file 7). year of exposure. Candidate V V-ATPase subunit-a in S. pistillata: gene Results 0 structure, amino acid sequence and phylogenetic analysis Candidate Na+-H+ exchanger (SLC9) in S. pistillata: gene As previously described for spiSLC9, we identified one structure, amino acid sequence and phylogenetic analysis gene homologous to human V V-ATPase subunit-a in We identified genes homologous to human SLC9 in the 0 the genome and transcriptome of S. pistillata. The gene genome and transcriptome of S. pistillata [46, 47]. and transcript information of the V V-ATPase subunit- Phylogenetic analysis of S. pistillata SLC9 proteins 0 a gene is given in Fig. 2a and Additional file 2. spiV V- (spiSLC9) with functionally characterized SLC9 in 0 ATPase subunit-a exists in four splice variants and is humans allowed us to group the corresponding S. pistil- one of the 14 subunits composing the spiV-ATPase lata genes within three subfamilies, namely, subfamilies (Additional file 8). A, B and C (Fig. 1). Members of subfamily A are distrib- The spiV V-ATPase subunit-a protein has six pre- uted in two different clusters (plasma membrane and or- 0 dicted TMs (Additional file 3) and exhibits similarity to ganelle) that contain plasma membrane and organelle human homologs (44–64% similarity and 59–76% iden- homologs, respectively. In addition, organelle homologs tity). Several residues (Fig. 2b) are conserved between include two distinct branches (A8 and A6/7). Of the spiV V-ATPase and hV V-ATPase, such as R735, seven SLC9 genes identified in S. pistillata, four belong 0 0 L739, H743, E789, L746, R799 and V803. Regarding to subfamily A (the NHE subfamily), one (spiSLC9A1)is post-translational modifications, several phosphorylation a plasma membrane homolog, two (spiSLC9A6 and and N-glycosylation sites were predicted for spiV V- spiSLC9A7) are A6/7 organelle homologs; one (spiSLC9A8) 0 ATPase subunit-a (Additional file 7). is an A8 organelle homolog; two (spiSLC9B1 and As previously described, we identified putative V V- spiSLC9B2) belong to subfamily B (the NHA subfamily); 0 ATPase subunit-a homologs in other species (Fig. 3). and one (spiSLC9C) belongs to subfamily C (the mamma- lianspermNHE-likesubfamily).Thegeneandtranscript + information of SLC9 family members is given in Add- Candidate voltage-gated H channels (HvCN) in S. itional files 1 and 2. pistillata: gene structures, amino acid sequences and The exchange domain of spiSLC9s is predicted to have phylogenetic analysis 8 to 12 transmembrane segments (TMs) (Additional file 3), Data mining in the genome and transcriptome of S. pis- which display sequence conservation in contrast to the tillata allowed us to identify two genes, spiHvCN1.1 and higher variability observed at the N-termini and C-termini spiHvCN1.2, homologous to human hHvCN1. The gene of the proteins. Sequence comparison of spiSLC9s showed and transcript information of the two spiHvCN genes is similarities between members of the same subfamily, and given in Fig. 4a and Additional file 2. Capasso et al. BMC Molecular and Cell Biology (2021) 22:18 Page 4 of 19

Fig. 1 A maximum likelihood phylogenetic (Phyml, LG + I + G) tree of human and cnidarian SLC9s (protein sequences were previously aligned by Clustal Omega). Phylogenetic analysis of S. pistillata SLC9 sequences with functionally characterized SLC9s in H. sapiens grouped cnidarian SLC9s within three different subfamilies: subfamily A, including plasma membrane and organelle homologs, which are represented in blue and red, respectively; subfamily B, which is represented in orange; and subfamily C, which is represented in yellow. Cnidarian species include Stylophora pistillata (Spi), Acropora digitifera (Adi), Nematostella vectensis (Nve), Aiptasia pallida (Apa), Corallium rubrum (Cru), Dendronephthya gigantea (Dgi), Amplexidiscus fenestrafer (Afe), and Discosoma sp. (Dsp). Chordata species include Homo sapiens (Hsa)

spiHvCN1.1 and spiHvCN1.2 share 35% identity and conserved residues relevant to HvCN activity mostly 62% similarity and possess four predicted transmem- located in the TMs. The two spiHvCN sequences brane domains (TMs) (Additional file 3). The TMs exhibit similarity with hHvCN1: spiHvCN1.1 has 40% displaysequenceconservation(Fig.4b), with highly identity and 67% similarity to hHvCN1, and Capasso et al. BMC Molecular and Cell Biology (2021) 22:18 Page 5 of 19

Fig. 2 a Exon/intron organization of V0 V-ATPase subunit a in the genome of S. pistillata. Exons are represented as boxes, whereas introns are depicted as lines. b Sequence comparison of S. pistillata and H. sapiens V0 V-ATPase subunit a. Identical and similar amino acids (aa) are shaded in black and grey, respectively. The boxes represent the predicted transmembrane segments in H. sapiens V0 V-ATPase subunit a. The circles and crosses represent + phosphorylation and N-glycosylation sites, respectively, in spiHvCN1.1 and spiHvCN1.2. The asterisk indicates a conserved R relevant to H transport

spiHvCN1.2 has 28% identity and 61% similarity to between spiHvCN1.1 and hHvCN1. Regarding post- hHvCN1. Several basic and acidic residues (R205, translational modifications, several phosphorylation R208, R211, H140, E153 and D174) are conserved be- sites were predicted for both spiHvCNs, whereas N- tween spiHvCNs and hHvCN1, whereas the residues glycosylation sites were predicted only for spiHvCN1.1 D112, D123, D185 and E119 are conserved only (Additional file 7). Capasso et al. BMC Molecular and Cell Biology (2021) 22:18 Page 6 of 19

Fig. 3 Maximum likelihood (Phyml, LG + I + G) phylogenetic tree of V0 V-ATPase subunit-a (protein sequences were previously aligned by Clustal Omega). Cnidarian species include Stylophora pistillata (Spi), Acropora digitifera (Adi), Nematostella vectensis (Nve), Aiptasia pallida (Apa), Corallium rubrum (Cru), Dendronephthya gigantea (Dgi), Amplexidiscus fenestrafer (Afe), and Discosoma sp. (Dsp). Mollusca species include Crassostrea gigas (Cgi). Echinodermata species include Strongylocentrotus purpuratus (Spu) and Acanthaster planci (Apl). Chordata species include Homo sapiens (Hsa) and Ciona intestinalis (Cin). Placozoa species include Trichoplax adhaerens (Tad). Porifera calcarea species include Sycon ciliatum (Sci). Porifera Homoscleromorpha species include Oscarella carmela (Oca)

Sequence similarity searches of available proteomic Tissue-specific gene expression of S. pistillata H+ and genomic datasets using the human HvCN1 protein transporters + identified putative HvCN homologs in several evolution- Quantitative real-time PCR was used to detect H trans- arily distant species (Fig. 5). Members of Hexacorallia porter mRNA expression (relative mRNA quantification, are the only species that possess two HvCNs, which Rq) in the oral fraction and the total colony (prepared splits the tree into two groups: HvCN1.1 and HvCN1.2. according to Ganot et al., 2015 and Zoccola et al., 2015) Capasso et al. BMC Molecular and Cell Biology (2021) 22:18 Page 7 of 19

Fig. 4 a Exon/intron organization of spiHvCNs in the genome of S. pistillata. Exons are represented as boxes, whereas introns are depicted as lines. b Sequence comparison of S. pistillata and H. sapiens HvCN proteins. Identical and similar amino acids (aa) are shaded in black and grey, respectively, whereas aa that are missing from the other sequence are denoted by dashes. The boxes represent the predicted transmembrane segments in human

HvCN1 (S1-S4). The circles and crosses represent phosphorylation and N-glycosylation sites, respectively, in spiHvCN1.1 and spiHvCN1.2

of S. pistillata. The results showed no differential ex- spiSLC9A-B, spiV0 V-ATPase subunit-a and spiHvCN pression of spiSLC9A1, spiSLC9B1, spiSLC9B2 or spiV0 genes was carried out in S. pistillata micro-colonies ex- V-ATPase subunit-a (Fig. 6a, e, f and g and Add- posed to control and acidified seawater (pH 8.1 and 7.2, itional file 9) between the two coral fractions. spiSLC9A6 respectively) for two different durations: 1 week and 1 and spiSLC9A7 (Fig. 6b and c) were more highly year. The results showed that there was no difference in expressed (p-value = 0.023 and 0.018, respectively) in the the expression of these genes between micro-colonies oral fraction than in the total colony, and spiSLC9A8 exposed to pH 8.1 and pH 7.2 for 1 week (Fig. 7 and (Fig. 6d) was more highly expressed (p-value = 0.108) in Additional file 10). After 1 year of exposure to lower pH, the total colony than in the oral fraction. Finally, there was no difference in the expression of most H+ spiHvCN1.1 (Fig. 6h) was more highly expressed (p- transporter-coding genes (Fig. 8 and Additional file 10). value = 0.107) in the total colony than in the oral frac- Only spiSLC9A1 was more highly expressed (p-value = tion, whereas spiHvCN1.2 (Fig. 6i) was more highly 0.029) at pH 7.2 than at the control pH of 8.1 expressed (p-value = 0.007) in the oral fraction than in (Additional file 10). the total colony. Discussion Effect of ocean acidification on the gene expression of S. Phylogeny, domain topology and motif analysis of SLC9s pistillata H+ transporters from S. pistillata To study the effect of seawater acidification on H+ trans- We identified SLC9 homologs in all anthozoan species porter mRNA expression, real-time qPCR analysis of the (Fig. 1). The anthozoan SLC9 proteins cluster within at Capasso et al. BMC Molecular and Cell Biology (2021) 22:18 Page 8 of 19

Fig. 5 Maximum likelihood (Phyml, LG + I + G) phylogenetic tree of voltage-gated proton channels (protein sequences were previously aligned by

Clustal Omega). HvCN1.1 and HvCN1.2 are separated into two semicircles. Cnidarian species include Stylophora pistillata (Spi), Acropora digitifera (Adi), Nematostella vectensis (Nve), Aiptasia pallida (Apa), Corallium rubrum (Cru), Dendronephthya gigantea (Dgi), Amplexidiscus fenestrafer (Afe), and Discosoma sp. (Dsp). Mollusca species include Crassostrea gigas (Cgi). Echinodermata species include Strongylocentrotus purpuratus (Spu) and Acanthaster planci (Apl). Chordata species include Homo sapiens (Hsa) and Ciona intestinalis (Cin). Placozoa species include Trichoplax adhaerens (Tad). Porifera calcarea species include Sycon ciliatum (Sci). Porifera Homoscleromorpha species include Oscarella carmela (Oca) least one subfamily (A, B and C), as previously described, and a variable cytosolic domain. In addition, several key suggesting a common ancestor at the time of Bilateria- residues important for the function of hSLC9s are also Radiata separation. conserved in spiSLC9s. For the A subfamily (Additional The spiSLC9s characterized in S. pistillata show simi- file 4), these residues include F161, which is important lar characteristics to those of human SLC9s [25, 48], for Na+ transport and acts as a pore-lining residue [49]; such as a highly homologous transmembrane domain P167 and P168, which play a structural role in the Capasso et al. BMC Molecular and Cell Biology (2021) 22:18 Page 9 of 19

Fig. 6 Relative mRNA quantification (Rq)ofSLC9s, V0 V-ATPase subunit-a and HvCNs measured in total (total colony) and oral (oral fraction) fractions. Box and whisker plots show the first, second (median) and third quartiles (horizontal lines of the boxes) and the respective whiskers (vertical lines spanning the lowest and highest data points of all data, including outliers). The replicate numbers (n = 3) represent separate coral samples. The asterisks and points indicate significant differences (• 0.11 ≤ p-value≤0.10 and ** p-value< 0.05) folding of the transmembrane domain [50] and influence within transmembrane structures and facilitate the for- the targeting and expression of SLC9A proteins [49]; mation of membrane pores. This feature might facilitate and R440 and G455/G456, which are located in the so- access to the mitochondrial inner membrane, where called “glycine rich region” and contribute to the proper these proteins are highly abundant [52]. Finally, the functioning of the putative “pHi sensor” domain [51]. members of subfamily C (Additional file 6) contain two For the B subfamily (Additional file 5), the conserved domains that are conserved with the human homolog: features between the human and S. pistillata homologs the VSD and the CNBD. The VSD of spiSLC9C carries are especially apparent between glycine zipper (GZ) se- positively charged residues (K and R) in S4 that are also quences. GZ sequences mediate close helix-helix folding strongly conserved in the SLC9C of Strongylocentrotus Capasso et al. BMC Molecular and Cell Biology (2021) 22:18 Page 10 of 19

Fig. 7 Relative mRNA quantification (Rq)ofSLC9s, V0 V-ATPase subunit-a and HvCNs at 1 week of pCO2 exposure plotted against pH 8.1 and 7.2 (n =5) purpuratus, Ciona intestinalis, Lepisosteus oculatus and properties with previously characterized SLC9s indicate Drosophila melanogaster [53]. Some of these residues that they retain similar functions and provide insights are missing in Homo sapiens, suggesting that voltage ac- into their activation mechanism and regulation in S. tivation differs between these species and humans. The pistillata. CNBD also regulates the activity of spiSLC9C, probably through the binding of cyclic adenosine monophosphate Phylogeny, domain topology and motif analysis of V0 V- (cAMP) produced by the soluble adenylyl cyclase en- ATPase subunit-a from S. pistillata zyme (sAC), as reported in the sea urchin [53]. We identified V0 V-ATPase subunit-a homologs in Overall, the conserved features of spiSLC9s align with (Fig. 3). In contrast to most species, which + + their roles as Na /H exchangers. Their shared possess more than one V0 V-ATPase subunit-a homolog, Capasso et al. BMC Molecular and Cell Biology (2021) 22:18 Page 11 of 19

Fig. 8 Relative mRNA quantification (Rq)ofSLC9s, V0 V-ATPase subunit-a and HvCNs at 1 year of pCO2 exposure plotted against pH 8.1 and 7.2 (n = 5). The asterisks indicate significant difference (** p < 0.05) anthozoans possess only one. This suggests that the allow H+ to translocate across the membrane [56]. Previ- specialization of V0 V-ATPase subunit-a homologs could ous studies in yeast allowed the identification of func- be phylum-specific or even species-specific [54]. tional residues in V0 V-ATPase subunit-a through the V0 V-ATPase subunit-a in S. pistillata shares similar use of random and site-direct mutagenesis. Among these characteristics with those in yeast and humans, including residues, R735 is known to play an essential role in pro- the number of predicted TMs, which falls within the ton transport, as mutations of this residue result in TM range of its homologs (5–8 TMs) [55]. TMs are complete inactivation of the ATP-dependent H+ trans- thought to form proton-conducting hemichannels that port of the V-ATPase [26]. The conservation of this Capasso et al. BMC Molecular and Cell Biology (2021) 22:18 Page 12 of 19

residue in S. pistillata suggests that R735 fulfils its role spiHvCN1.2. This could result in a different sensitivity to + in H transport (Fig. 2b). Other residues (L739, H743, voltage, with spiHvCN1.2 being less sensitive than L746, E789, R799 and V803) involved in proton trans- spiHvCN1.1. Additionally, among these residues, D112 location and ATPase activity [55] are also conserved in in S3, which is known to be essential for proton and S. pistillata. charge selectivity [61], is missing and replaced by E112 Overall, the features conserved between spiV0 V- in spiHvCN1.2. However, a previous study demonstrated ATPase subunit-a and its human and yeast homologs that the substitution of D112 with an acidic residue in align with its role as the subunit-a of the V0 V-ATPase. the same position maintained proton specificity in Furthermore, the identification of homologs of all the V- hHvCN1 [61], suggesting that both spiHvCNs are proton ATPase subunit-encoding genes in the genome and specific. Another difference concerns the Zn+2-binding transcriptome of S. pistillata (Additional file 8) suggests residues of spiHvCNs. In humans and mice, proton cur- a conserved organization between the S. pistillata V- rents are suppressed by extracellular Zn+2, which binds ATPase and the human V-ATPase. to four Zn+2-coordinating residues (E119, D123, H140 and H193) [64, 65]. By sequence comparison, we ob-

Phylogeny, domain topology and motif analysis of HvCNs served that at these positions, some residues are not from S. pistillata conserved in spiHvCN1.1 (H193G) and are even less One HvCN family member (HvCN1.1) was identified in conserved in spiHvCN1.2 (E119L, D123E and H193K). all species examined in this study, including the four Hence, we suggest that the replacement of Zn+2-coord- anthozoan orders, namely, Actinaria, Alcyonacea, Coral- inating residues with other residues potentially affects +2 limorpharia and (Fig. 5). In addition, we re- the Zn sensitivity of spiHvCNs, as reported in verte- +2 port for the first time a second member of the HvCN brates [66, 67]. Since spiHvCN1.1 contains more Zn - family (HvCN1.2) in some . Genomic and tran- coordinating residues, we propose that it is more sensi- +2 scriptomic searches of HvCN1.2 in public databases of tive to Zn than spiHvCN1.2. Octocorallia and Hydrozoa (Hydra magnipapillata) did Overall, the conserved features of the spiHvCNs align not produce any results (data not shown). The selective with their roles as voltage gated H+-channels. Common expression of HvCN1.2 only in Hexacorallia suggests properties between the spiHvCNs and hHvCN preserve that it is specific to this cnidarian subclass, and its pres- their voltage sensitivity and proton specificity, with some ence in noncalcifying anthozoans (Corallimorphs and differences concerning their Zn+2 sensitivity. In addition, Actinaria) suggests that it is not linked to the appear- distinctive properties between different spiHvCNs sug- ance of aragonite biomineralization in Scleractinia [57]. gest differential regulation, possibly linked to their The two voltage-gated proton channels (spiHvCNs) localization/function. Future analyses, however, are re- characterized in S. pistillata are highly divergent. This quired to validate these assumptions and provide insight finding is further supported by the length of the phylo- into their physiological role. genetic branch (Fig. 5) that separates the two homologs: + the amino acid similarity between spiHvCN1.1 and Tissue-specific expression patterns of H transporter hHvCN1.1 is higher than that between spiHvCN1.1 and genes in S. pistillata spiHvCN1.2. Whole coral colonies are typically used in conventional The spiHvCNs possess molecular properties that are techniques of gene expression analysis, limiting the pos- hallmarks of all HvCNs, such as the four transmembrane sibility of further differentiating specific gene expression segments, the basic (R) and acidic residues (D and E) as- between oral and aboral tissues. These tissues contain sociated with voltage sensing, and the coiled-coil struc- different cell subtypes; some of them are more abundant ture at the C-terminus [58]. The gating of proton (e.g., endosymbiotic dinoflagellates) or exclusively found channels is tightly regulated by pH and voltage, ensuring (e.g., cells specialized for food digestion and that they open only to extrude H+ from the cell [43]. reproduction, nematocysts) in the oral tissue, whereas Phosphorylation of the spiHvCNs might activate these others are exclusively found in the aboral tissue (e.g., channels and enable them to open faster and at less calcifying cells) (Veron et al., 1993; Peter et al., 1997) positive voltages than those required without activation, [68]. As several physiological functions are associated as previously reported in human leukocytes [59–62]. with these cellular subtypes, analysing the differential + SpiHvCN1.1 and spiHvCN1.2 share similar structures gene expression of H transporters in the two coral tis- and organizations (Fig. 4). However, differences identi- sues helps identify the potential physiological processes fied at the protein sequence level might reflect some in which they might participate. unique features. First, many acidic residues, which are To perform this task, we used a previously developed known to be associated with voltage sensing in hHvCN1 micro-dissection protocol [20, 69] to separate the oral [58, 63], are present in spiHvCN1.1 but not in fraction (including the oral disc and most of the Capasso et al. BMC Molecular and Cell Biology (2021) 22:18 Page 13 of 19

body, with no or minimal contamination of cells from ATPase subunit-a homolog is ubiquitously expressed the aboral tissue) from the total colony. We then com- and differs from those identified in humans. Indeed, the + pared the expression of H transporters in the oral frac- human V0 V-ATPase subunit-a isoforms have different tion to that in the total colony. tissue distributions with intracellular or apical/basolat- Our results demonstrate that some H+ transporters eral membrane localization [31, 55, 86–89]. This differ- are more highly expressed in the oral fraction than in ence is probably linked to the primers used for the the total colony (hereafter referred to as “oral-specific”); detection of the coral V0 V-ATPase subunit-a, which do others are more highly expressed in the total colony not discern the different isoforms (X1, X2, X3 and X4; than in the oral fraction (hereafter referred to as “aboral- see Additional file 8) but recognize all of them. We sug- specific”); and others are expressed at the same levels in gest that other ubiquitous transporters play housekeep- both fractions (hereafter referred to as “ubiquitous”) ing roles. spiSLC9A1, for example, could play a role in (Fig. 6). Using H+ transporter tissue expression and the homeostatic pH regulation on the basolateral cell mem- existing literature in other systems as supporting infor- brane, and spiSLC9B1–2 might participate in organismal mation, we discuss the role that these transporters might ion homeostasis on the mitochondrial inner membrane, play in corals. as reported in vertebrates [19, 90–93]. Interestingly, The oral-specific H+ transporters include spiSLC9A6, spiSLC9A1 was the only transporter affected by seawater spiSLC9A7 and spiHvCN1.2. spiSLC9A6 and spiSLC9A7 acidification after 1 year of exposure (Fig. 8). Its activa- are organellar homologs (Fig. 1), and as reported for tion might be triggered by the pHi sensor domain (see humans [48, 70], they might play a role in vesicular Discussion 3.1.1), which activates the transporter at low neurotransmitter uptake in oral polyps, where an elabor- pHi values, similar to humans [19]. Figure 9 summarizes ate nerve ring system is present [71]. In addition, cells in the tissue distribution of the principal acid-base trans- the oral tissue are enriched with zooxanthellae [72], porters that could participate in the intracellular pH which produce and incorporate high levels of anionic regulation of coral cells based on the results obtained by superoxide (e.g., throughout photosynthesis) and Zn+2 performing real-time PCR of oral fraction and total col- (e.g., through uptake from seawater) that accumulate in ony coral samples. These transporters include the H+ the host cytoplasm [73–75]. Thus, spiHvCN1.2 might transporters SLC9, V-ATPase and HvCN, which were + − favour the exit of H at the basolateral membrane of characterized in the present study, and the HCO3 trans- these cells, as reported in human osteoblasts [76], pre- porters SLC4 and SLC26, which were characterized by venting the membrane depolarization to extreme nega- Zoccola et al. in 2015. − tive voltages associated with O2 electron transfer. Additionally, the decreased Zn+2 sensitivity of Conclusions spiHvCN1.2 compared to that of spiHvCN1.1 (see Dis- This study provides the first molecular characterization cussion 3.1.3) could be correlated with the higher Zn+2 in the coral S. pistillata of several families of H+ trans- concentration levels in the oral (symbiotic) cells [67, 77– porters, which are known as pHi regulators in animal 80]. cells. Most importantly, we report for the first time a + The aboral-specific H transporters are spiHvCN1.1 novel member of the HvCN gene family, HvCN1.2, in and spiSLC9A8. These transporters could play a role in the cnidarian subclass Hexacorallia. The identification of + the pHi regulation of calcifying cells. During the calcifi- conserved residues between coral and human H trans- cation reaction, H+ is produced in the ECM and needs porter homologs suggests functional conservation re- to be removed to promote an alkaline environment lated to intracellular pHi regulation. favourable for calcification [11, 81]. The Ca+2-ATPase at However, additional experiments (e.g., gene silencing the apical side of the calcifying cells has been suggested experiments, pharmacological experiments using inhibi- to be involved in pumping out H+ from the ECM [41]. tors, electrophysiological measurements of H+ currents, On the basal side of the cell membrane, spiHvCN1.1 etc.) with a larger number of replicates need to be car- might contribute to extruding excess H+ from the cyto- ried out in the future to demonstrate the participation of plasm of calcifying cells, similar to the role it plays in these H+ transporters in coral acid-base cellular homeo- coccolithophores [82]. Finally, the organellar spiSLC9A8 stasis. Moreover, we assessed the tissue specificity of the might regulate medial/trans-Golgi pH and intracellular H+ transporter gene families in the coral S. pistillata, trafficking, as in humans [83, 84], since in calcifying and we observed that their expression was not restricted cells, this function is particularly necessary for the regu- to only one specific tissue (oral or aboral), as reported − lation of organic matrix synthesis and secretion [85]. for some members of the HCO3 gene family [20]. How- + The rest of the H transporters (V0 V-ATPase ever, we observed higher or lower expression profiles in subunit-a, spiSLC9A1, spiSLC9B1 and spiSLC9B2) are the oral or aboral tissues. These results both highlight + ubiquitous. Our results suggest that the coral V0 V- the importance of H transporters in the coral colony Capasso et al. BMC Molecular and Cell Biology (2021) 22:18 Page 14 of 19

Fig. 9 (See legend on next page.) Capasso et al. BMC Molecular and Cell Biology (2021) 22:18 Page 15 of 19

(See figure on previous page.) Fig. 9 Model of acid-base transporters involved in intracellular pH regulation expressed on the apical (AM) and basolateral (BLM) membranes of coral cells throughout the tissue layers. The roles of other ion channels and transporters involved in other cellular processes are not considered here. Transporters that are more highly expressed in the oral fraction (oral-specific) are coloured in blue, those that are more highly expressed in the total colony (aboral-specific) are coloured in orange, and those that are expressed at the same levels in both fractions (ubiquitous) are coloured in green. Other enzymes (CA = carbonic anhydrase) and transporters (PMCA = Ca+2 ATPase) involved in the H+ flux balance are represented in bold letters

and suggest that they take part in homeostatic (e.g., intra- chemistry was manipulated by bubbling with CO2 to re- cellular acid-base balance) and physiological processes duce the pH from the control value of pH 8.1 to the tar- (e.g., calcification, photosynthesis, food digestion). Finally, get value of 7.2. Twenty coral nubbins were randomly we investigated the impact of OA on H+ transporter gene distributed among four experimental tanks (n = 5 for expression in S. pistillata, and we identified one candidate each experimental tank): two tanks (pH 8.1 and 7.2) were gene (spiSLC9A1) involved in the coral response to ocean reserved for 1 week of exposure, and two were reserved acidification that showed differential expression after for 1 year of exposure. The experiments were repeated long-term exposure to acidified seawater (1 year). The three times to ensure reliability. Similar results were ob- other H+ transporters did not show any significant tained for each experiment (not shown). changes at the gene level under seawater acidification con- ditions. Nevertheless, the modulation of gene expression Data mining can also occur at the protein level, which should be inves- The amino acid sequences of human H+ transporters tigated in future studies. The influence of other environ- were retrieved from NCBI (http://www.ncbi.nlm.nih.gov/ mental factors, e.g., temperature, remains to be tested and protein) and used as a bait to mine (BLAST) the tran- will enrich the understanding of coral phenotypic plasti- scriptome, genome and EST databases of the following city. This knowledge is especially relevant to our under- phyla: Cnidaria (Stylophora pistillata, Acropora digiti- standing of the ability of benthic animals to buffer the fera, Nematostella vectensis, Aiptasia pallida, Amplexi- impacts of environmental changes, thereby providing discus fenestrafer, Discosoma sp., Corallium rubrum and more time for genetic adaptation to occur. In addition, re- Dendronephthya gigantea), Mollusca (Crassostrea gigas), sponses to environmental changes are often species- Echinodermata (Strongylocentrotus purpuratus), Chord- specific [9], with physiological differences that can poten- ata (Ciona intestinalis), Porifera (Sycon ciliatum) and tially reflect a different profile of H+ transporter gene ex- Placozoa (Trichoplax adhaerens). The following web pression, which could also be investigated in other coral servers were used: NCBI (http://www.ncbi.nlm.nih.gov/), species. Such comparative studies might aid in the devel- EnsemblMetazoa (https://metazoa.ensembl.org/), Reef- opment of molecular markers linked to pHi tolerance Genomics (http://reefgenomics.org) and Cnidarian Data- traits in coral reef populations. base (http://data.centrescientifique.mc/).

Methods Sequence analysis Biological materials Putative transmembrane helices in proteins were pre- Experiments were conducted on the symbiotic scleractinian dicted by the Center for Biological Sequence Analysis coral Stylophora pistillata grown in the long-term culture Prediction Server (http://www.cbs.dtu.dk/services/) using facilities at the Centre Scientifique de Monaco in aquaria the TMHMM algorithm. On the same platform, phos- supplied with seawater from the Mediterranean Sea (ex- phorylation and N-glycosylation prediction analyses change rate 2% h − 1) under the following controlled condi- were performed using NetPhos and NetNGlyc. tions: semi-open circuit, temperature of 25 °C, salinity of − − 38, light exposure of 200 μmol photons m 2 s 1,anda12 Phylogenetic analyses h:12 h light:dark cycle. Samples were prepared from one Alignment of H+ transporter amino acid sequences was mother colony as nubbins suspended on monofilament performed using Clustal Omega on the EMBL-EBI ser- threads. After 3 weeks of cicatrisation, the first set of sam- ver (https://www.ebi.ac.uk/). Based upon the produced ples (n = 3) was used for the oral fraction micro-dissection amino acid alignments, maximum likelihood estimates experiment, and another set (n =20)wasusedfortheex- of the topology and the branch length were obtained posure to seawater acidification experiment. using PhyML v3.1 [96]. The model of substitution used in this step was LG + G, which was previously selected Exposure to seawater acidification over others via alignment analysis with ProtTest3.4.2 The seawater acidification setup was performed as de- [97]. Phylogenetic trees were then edited using FigTree scribed previously [9, 94, 95]. Briefly, carbonate v1.4.4 (Rambaut et al., 2014). Capasso et al. BMC Molecular and Cell Biology (2021) 22:18 Page 16 of 19

Oral fraction micro-dissection Abbreviations Coral micro-dissection was performed as indicated pre- CNBD: Cyclic nucleotide-binding domain; CPA1: Cation proton antiporter 1; CPA2: Cation proton antiporter 2; ECM: Extracellular calcifying medium; viously [20, 69]. Briefly, coral nubbins (n = 3) were set to GZ: Glycine zipper; HvCN: Voltage-gated proton channel; NaT-DC: Na- rest in a glass petri dish filled with seawater and tricaine transporting carboxylic acid decarboxylase; OA: Ocean acidification; mesylate. Once polyps were extended, oral fractions (in- sAC: Soluble adenylyl cyclase; SLC9: Solute carrier 9; TM: Transmembrane segment; V-ATPase: Vacuolar-type electrogenic H+-ATP hydrolase; cluding the oral disc and most of the polyp body) were VSD: Voltage-sensing domain cut from the coral colony (total colony) using micro- dissection scissors under a binocular microscope. Both Supplementary Information fractions (oral fraction and total colony) were then used The online version contains supplementary material available at https://doi. for RNA extraction. org/10.1186/s12860-021-00353-x.

Additional file 1. Exon/intron organization of SLC9s in the genome of S. pistillata. Real-time PCR (qPCR) Additional file 2. Gene and transcript information of H+ transporter Total RNA extraction and cDNA synthesis were per- family members in S. pistillata. formed as described previously [98]. Briefly, RNA was Additional file 3. Transmembrane segment prediction (TMs) of H+ isolated from biological triplicates that underwent transporter proteins in S. pistillata. micro-dissection or from quintuplicates collected for Additional file 4. Sequence comparison of the H. sapiens SLC9A1 and S. each pH treatment using an RNeasy kit (Qiagen) accord- pistillata SLC9A proteins. ing to the manufacturer’s instructions. Reverse transcrip- Additional file 5. Sequence comparison of the S. pistillata and H. sapiens SLC9B proteins. tion was then performed using SuperScript IV Reverse μ Additional file 6. Sequence comparison of the S. pistillata and H. Transcriptase (Invitrogen) on 2 g of RNA. Thermo cycler sapiens SLC9C1 proteins. The boxes represent human voltage-sensing do- conditions were set as follows: 50 min at 50 °C, 7 min at mains (S1-S4), and the asterisks indicate conserved positively and nega- 25 °C, 50 min at 50 °C and 5 min at 85 °C. qPCR runs were tively charged residues relevant to voltage sensing. The rectangles indicate conserved positively charged residues in S4 that are present in S. performed in 96-well plates on a QuantStudio 3 (Applied pistillata but missing in hSLC9C1. The triangles indicate residues involved Biosystems) machine using PowerUpTM SYBRTM Green in the cyclic nucleotide-binding domain. Master Mix for PCR amplification. The primer sequences Additional file 7. Number of phosphorylation sites and N-glycosylation used are provided in Additional file 11. Relative expression sites predicted for S. pistillata H+ transporters. was calculated using Biogazelle qBase + 2.6TM [99]. Gene Additional file 8. A. V0 V-ATPase subunit-a isoforms in coral S. pistillata. B. Human and coral homologs of V-ATPase subunits. expression was normalized (relative mRNA quantification, Additional file 9. Statistical analysis of the relative mRNA quantification Rq) to the expression of two reference genes, ubiquitin- (Rq)ofSLC9s, V0 V-ATPase subunit-a and HvCNs (N =3)inS. pistillata total 60S ribosomal protein (L40) [100] and an acidic ribosomal (total colony) and oral (oral fraction) fractions. phosphoprotein P0 (36B4) [101], after these genes were Additional file 10. Statistical analysis of the relative mRNA determined to have acceptably low M values and coeffi- quantification (Rq)ofSLC9s, V0 V-ATPase subunit-a and HvCNs (n =5)at pH 8.1 and 7.2 after 1 week and 1 year of pCO2 exposure. The asterisks cients of variation [99]. indicate significant differences (** p < 0.05). Additional file 11. List of S. pistillata H+ transporter genes with RT-PCR primers and product sizes. Statistical analysis Statistical analysis was performed using R v3.5.2 software. Acknowledgements For the oral fraction micro-dissection experiment, a fixed We would like to thank Dominique Desgré for coral maintenance and number of samples (n = 3) was used due to technical limi- Alexander Venn for kindly revising the text of the manuscript. This study was conducted as part of the Centre Scientifique de Monaco Research Program, tations. For the exposure to seawater acidification experi- which is supported by the Government of the Principality of Monaco. ment, a sample size estimation was performed, and n =5 samples were used. For both experiments, the normal dis- Authors’ contributions ’ ST and DZ designed and conceived the study. LC conducted the study. LC, tribution of the data was evaluated using Shapiro-Wilk s PG, VPB and DZ analysed the data. LC, ST and DZ wrote the manuscript. All test. Samples with a standard deviation ≥5wereexcluded authors read and approved the manuscript. from the analysis. This was the case for spiSLC9C,whose Funding qPCR results showed great variability, probably due to its Funding of this study was provided by the Government of the Principality of extremely low expression values. A t-test was used to Monaco. The funding body played no role in the design, collection, analysis, identify differentially expressed genes between S. pistillata or interpretation of the data; the writing of the manuscript; or the decision fractions (oral fraction and total colony) and pH treat- to submit the manuscript for publication. ments (pH 8.1 and pH 7.2). We considered 0.10 ≤ p-values Availability of data and materials ≤0.11to indicate near-marginal significance (•); p-values All data needed to evaluate the conclusions in the paper are present in the ≤0.1 as significant (**); and p-values ≤0.05 as highly signifi- manuscript and/or the Additional Files. Additional data related to this manuscript are available from the corresponding author on reasonable cant (*). request. Genomic and transcriptomic data were obtained from the publicly Capasso et al. BMC Molecular and Cell Biology (2021) 22:18 Page 17 of 19

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